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Abstract:

The present disclosure relates to the identification of a QTL associated
with high ethanol tolerance in Saccharomyces spp. More specifically, it
relates to specific alleles of MKT1 and APJ1 possibly combined with a
specific allele of SWS2 that are important in obtaining a high ethanol
tolerance in Saccharomyces spp. It relates further to the use of such
alleles in the construction of high ethanol tolerant strains, and the use
of these alleles in screening for ethanol tolerance.

Claims:

1. A method of obtaining ethanol tolerance in a yeast, the method
comprising: utilizing an inactivated APJ1 allele to obtain ethanol
tolerance in the yeast.

2. The method according to claim 1, wherein said inactivated APJ1 allele
is a deletion mutant.

3. The method according to claim 1, wherein said allele is combined with
the expression of a mutant MKT1 allele.

4. The method according to claim 3, wherein said mutant MKT1 is
characterized by a glycine at position 30 and an arginine at position
453, or equivalent positions in homologous sequences.

13. The method according to claim 10, wherein the allele is combined with
expressing a mutant MKT1 allele.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a national phase entry under 35 U.S.C.
§371 of International Patent Application PCT/EP2012/061823, filed
Jun. 20, 2012, designating the United States of America and published in
English as International Patent Publication WO 2012/175552 A1 on Dec. 27,
2012, which claims the benefit under Article 8 of the Patent Cooperation
Treaty to European Application Serial No. 11170692.5, filed Jun. 21,
2011.

TECHNICAL FIELD

[0002] The present disclosure relates to the identification of a QTL
associated with high ethanol tolerance in Saccharomyces spp. More
specifically, it relates to specific alleles of MKT1 and APJ1 possibly
combined with a specific allele of SWS2 that are important in obtaining a
high ethanol tolerance in Saccharomyces spp. It relates further to the
use of such alleles in the construction of high ethanol tolerant strains,
and the use of these alleles in screening for ethanol tolerance.

BACKGROUND

[0003] Genetic analysis of polygenic, quantitative traits remains an
important challenge. It requires reliable scoring of many genetic markers
covering the whole genome. In yeast, the first successful approaches to
simultaneously map multiple genetic loci, that were either independent
(Winzeler et al., 1998) or involved in a quantitative trait (QTL)
Steinmetz et al., 2002), made use of SNP markers that were scored by
hybridization of genomic DNA from individual segregants to a gene
expression micro-array. Subsequently, a similar approach was used to map
QTL involved in traits such as sporulation efficiency (Deutschbauer and
Davies, 2005), gene expression (Brew et al., 2002), acetic acid
production (Marullo et al., 2007), cell morphology (Nogami et al., 2007)
and resistance to small-molecule drugs (Perlstein et al., 2007).

[0004] The advent of high-throughput sequencing technologies provides a
new way to score large numbers of SNPs as genetic markers. Application to
individual segregants remains cumbersome because of the high costs
involved. On the other hand, bulked segregant analysis was shown to be
efficient in identifying markers linked to specific genes (Michelmore et
al., 1991) and is robust to occasional phenotyping mistakes (Segre et
al., 2006). Schneeberger et al., (2009) showed that this approach worked
for a single mutation. They crossed an Arabidopsis thaliana mutant with
an unrelated strain, pooled 500 segregants with the mutant phenotype and
used the nucleotide frequency of SNPs detected by Illumina whole genome
sequence analysis in the DNA extracted from the pool to map the locus
with the mutation. Recently, Arnold et al., (2011) used a similar
approach to identify a single mutation responsible for a renal disease in
mice and Birkeland et al., (2010) to map a mutation causing a defect in
vacuole inheritance in S. cerevisiae. It has been suggested that in
principle this approach should also allow the simultaneous mapping of
multiple QTL present throughout the genome (Schneeberger et al., 2009;
Birkeland et al., 2010; Lister et al., 2009). However, to the best of our
knowledge, this has not been demonstrated yet for a typical quantitative
trait.

BRIEF SUMMARY

[0005] For this disclosure, we applied "pooled-segregant whole genome
sequence analysis" for the mapping of QTL involved in tolerance to high
ethanol levels (16-17%) in yeast. High ethanol tolerance is an exquisite
characteristic of the yeast Saccharomyces cerevisiae and is of prime
importance to the yeast fermentation industries (bioethanol, beer, wine
and other alcoholic beverages). Up to now, ethanol tolerance in yeast has
been studied mostly in laboratory yeast strains and always with low to
moderately high ethanol concentrations (5-12%). These studies have
revealed that properties like membrane lipid composition, chaperone
protein expression, and trehalose content are important determinants of
ethanol tolerance (D'Amore and Stewart, 1987; Ding et al., 2009).
Genome-wide transcriptomics and screening of deletion mutants have
revealed many genes required for tolerance to low/moderate ethanol
concentrations Fujita et al., 2006; Lewis et al., 2010; van Voorst et
al., 2006). In most of these studies, ethanol tolerance was determined
based on growth in the presence of ethanol. Furthermore, a genetic
dissection of ethanol tolerance was made in laboratory strains in which
ethanol tolerance was measured as survival after treatment with different
concentrations of ethanol under non-growing conditions. Short Tandem
Repeats (STRs) and Single Nucleotide Polymorphisms (SNPs), detected by
multiplex PCR, were used as genome-wide genetic markers and five QTL were
identified which explained about 50% of the phenotypic variation (Hu et
al., 2007). In contrast, nothing is known about the genetic loci or gene
polymorphisms that are responsible for the much higher ethanol tolerance
during growth of natural and industrial yeast strains compared to
laboratory strains.

[0006] Surprisingly, we found that "pooled-segregant whole genome sequence
analysis" can be used for mapping of QTL in yeast. Even more
surprisingly, we have identified and validated three genetic loci in a
Brazilian bioethanol production strain that are responsible for tolerance
to high ethanol levels during growth. In addition, we have dissected the
locus with the strongest linkage and identified two novel alleles with a
previously unrecognized, positive function in ethanol tolerance. The
locus also contained a mutant allele with a negative contribution to high
ethanol tolerance, which was located in between the two genes with a
positive contribution.

[0007] A first aspect of the disclosure is the use of an inactivated APJ1
(SEQ ID NO:2, accession number: genbank NP--014322 version
NP--014322.1, 26 Apr. 2011) allele, or a homologue, orthologue or
paralogue thereof, to obtain ethanol tolerance in yeast. Inactivated, as
used here, means that the expression can be lowered by mutations in the
promoter region, or that mutants in the open reading frame may occur,
affecting the biological activity of the gene. A "homologue," as used
here, encompasses a gene encoding a protein having amino acid
substitutions, deletions and/or insertions relative to the unmodified
protein in question and having similar biological and functional activity
as the unmodified protein from which it is derived. "Orthologue" and
"paralogue" encompass evolutionary concepts used to describe the
ancestral relationships of genes. Paralogues are genes within the same
species that have originated through duplication of an ancestral gene;
orthologues are genes from different organisms that have originated
through speciation, and are also derived from a common ancestral gene.
Preferably, the homologue, orthologue or paralogue shows at least 40%
identities at protein level, as measured by a BLASTp alignment (Altschul
et al., 1997; Altschul et al., 2005). Even more preferably, it has at
least 45%, more preferably 50%, more preferably 55%, more preferably 60%,
more preferably 65%, more preferably 70%, more preferably 75%, more
preferably 80%, more preferably 85%, more preferably 90%, most preferably
95% identities. Preferably, the inactivated allele is a disrupted or
deleted APJ1 mutant, including the complete deletion of the gene. The use
of an inactivated allele, as used here, means that in a haploid strain
the APJ1 gene is replaced by the inactivated allele, and in a diploid or
polyploidy or aneuploid yeast strain, at least one copy of the APJ1 gene
is replaced by the inactivated allele. Preferably, several copies are
replaced; most preferably all copies are replaced by the inactivated
allele. Ethanol tolerance, as used here, means that the strain, carrying
the inactivated allele can be grown at higher ethanol concentrations than
the parental strain. Preferably, ethanol tolerance means that the stain
is capable to grow on plates with at least 12% ethanol, preferably on
plates with at least 14% ethanol, more preferably on plates with at least
16%, more preferably at least 17%, most preferably at least 18% ethanol.
A "yeast," as used here, can be any unicellular fungus. Preferably, the
yeast is a species selected from the genera Saccharomyces,
Zygosaccharomyces, Brettanomyces, Kluyveromyces, Pichia, Pachysolen and
Candida. Preferably, the yeast is a brewers', wine or distillers yeast
selected from the genera Saccharomyces, Zygosaccharomyces and
Brettanomyces. Most preferably, the yeast is a Saccharomyces spp,
preferably Saccharomyces cerevisiae.

[0008] In one preferred embodiment, the use of the inactivated APJ1 allele
is combined with the use of a mutant MKT1 allele. The use of a mutant
MKT1 allele, as used here, means that in a haploid strain the MKTJ gene
is replaced by the mutant allele, and in a diploid or polyploidy or
aneuploid yeast strain, at least one copy of the MKT1 gene is replaced by
the mutant allele. Preferably, several copies are replaced; most
preferably all copies are replaced by the mutant. A mutant MKT1 gene is a
gene that encodes a protein that is different from the reference protein
(SEQ ID NO:3, Genbank accession number CAA95961, version CAA95961.1 dated
11 Aug. 1997). Preferably, the mutant encodes a protein carrying mutation
at positions 30 and 453, more preferably, the mutant encodes a protein
that has a glycine at position 30 and an arginine at position 453, most
preferably, the mutant encodes a protein that comprises, preferably
consists of SEQ ID NO:1 (table 3). Alternatively, the mutant MKT1 allele
is a mutant of a homologue, orthologue of paralogue (as defined earlier)
of the gene encoding MKT1.

[0009] In another preferred embodiment, the use of the inactivated APJ1
allele is combined with the overexpression of a wild type SWS2 gene. A
wild type SWS2 gene is a gene encoding a sws2p as given by SEQ ID NO:4
(Genbank accession number NP--014318, version NP--014318.1
dated 26 Apr. 2011) or a homologue, orthologue or paralogue thereof, as
defined above. Overexpression, as used here, means that the level of SWS2
protein in the strain carrying the inactivated APJ1 is higher than in the
parental strain. As a non-limiting example, overexpression can be
obtained by placing the coding sequence under control of a strong
promoter, or by increasing the copy number of the gene.

[0010] It is clear for the person skilled in the art that the inactivated
APJ1 allele can be combined with both overexpression of the wild type
SWS2 gene, as well as the expression of a mutant MKT1 allele, according
to the disclosure. Moreover, other ethanol tolerance improving genes can
be used to increase the effect of the inactivated APJ1 allele, whether in
combination with wild type SWS2 and/or mutant MKT1 or not.

[0011] Alternatively, the expression of mutant MKT1 allele, according to
the disclosure, or overexpression of the wild type SWS2 gene may be used
alone to obtain ethanol tolerance, or the combination of the expression
of mutant MKT1 allele, according to the disclosure, with overexpression
of the wild type SWS2 gene can be used to obtain ethanol tolerance.

[0012] Another aspect of the disclosure is a method for screening ethanol
resistant yeast, the method comprising the identification of
downregulating mutations in the APJ1 gene and/or the determination of the
G30 and/or R453 mutation in the Mkt1p. The APJ1 gene, as used here,
includes the promoter and teiniinator region. Downregulating mutations
are known to the person skilled in the art and include, but are not
limited to, insertions, deletions of premature stops in the coding
sequence. Determination of G30 and/or R453 mutation in the Mkt1p can be
carried out at protein level or at nucleic acid level; preferably, it is
carried out at nucleic level, by checking the coding sequence.

BRIEF DESCRIPTION OF THE FIGURES

[0013] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office upon
request and payment of the necessary fee.

[0014] FIG. 1: Ethanol tolerance of the Brazilian bioethanol production
strain VR1 and its segregant VR1-5B.

[0015] The ethanol tolerance of VR1 (diploid) and VR1-5B (haploid) was
determined by scoring growth of tenfold dilutions on YP plates with
different concentrations of ethanol. Both strains, as well as the
heterozygous VR1-5B/BY4741 strain (diploid), showed a clearly higher
ethanol tolerance than the control laboratory strains BY4741 (haploid)
and BY (diploid), which was obtained by crossing BY4741 with BY4742.

[0017] (A) QTL mapping by whole-genome sequence analysis of DNA extracted
from a pool of 136 segregants tolerant to at least 16% ethanol (Pool 1).
The genomic DNA of the parents, VR1-5B and BY4741, and the pool was
sequenced and aligned to identify SNPs. The nucleotide frequency of
quality-selected SNPs in the sequence of the pool was plotted against the
chromosomal position. Significant deviations from the average of 0.5
indicate candidate QTL linked to high ethanol tolerance. Upward
deviations indicate linkage to QTL in the ethanol tolerant parent VR1-5B.

[0018] (B) Application of a more stringent selection condition reveals
candidate minor loci determining high ethanol tolerance. For a locus on
chromosome II and XV, the SNP frequencies in the pool of segregants
tolerant to at least 17% ethanol (red line) show a more pronounced
deviation from random segregation in comparison to the pool of segregants
tolerant to at least 16% ethanol (green line). The difference in SNP
frequency between the two pools is certainly significant when the
confidence intervals do not overlap.

[0020] The genomic DNA of the parents, VR1-5B and BY4741, and the pool was
sequenced and aligned to identify SNPs. The nucleotide frequency of
quality-selected SNPs in the sequence of the pool was plotted against the
chromosomal position. Significant deviations from the average of 0.5
indicate candidate QTL linked to high ethanol tolerance. Upward
deviations indicate linkage to QTL in the ethanol tolerant parent VR1-5B.

[0022] The two major QTL on chromosomes V and XIV are not significantly
different between the two pools. However, in several instances, e.g., on
chromosomes II, XII and XV, minor loci can be identified showing a
significant difference between the two pools. These candidate QTL are
more distinctive in Pool 2 (17% ethanol) compared to Pool 1 (16%
ethanol). The difference in SNP frequency between the two pools is
certainly significant when the simultaneous confidence bands do not
overlap.

[0023] FIG. 5: Detailed statistics of the two major loci linked to high
ethanol tolerance.

[0024] The tables show for each marker in the two mapped major loci (A:
Chromosome V, B: chromosome XIV) the position of the marker, the number
of segregants in which the marker was scored, the association percentage
and the P value. The association percentage represents the percentage of
segregants with VR1-5B inheritance, i.e., the nucleotide from VR1-5B. The
markers with the strongest link are shown in bold.

[0025] FIG. 6: Fine-mapping and identification of the causative genes in
QTL3.

[0026] (A) The 87 kb locus defined by SNP markers S67, S68 and S69 in QTL3
showed the lowest probability of random segregation in 101 highly ethanol
tolerant segregants. Further fine-mapping was achieved by scoring five
additional markers within the 87 kb interval in the same segregants.
Calculation of the P values revealed the strongest link for a 16 kb locus
defined by markers S68, S68-1 and S68-2.

[0027] (B) The name and location of each ORF in the fine-mapped locus is
shown as annotated in SGD 20. The interval from nucleotide 466,599 till
485,809 was sequenced in VR1-5B and BY4741, which revealed 115
polymorphisms, of which part were in intergenic regions (numbers between
brackets). For the ORFs, only polymorphisms that change the amino acid
sequence are indicated (amino acid in BY4741, followed by position in the
protein and amino acid in VR1-5B). SAL1 has a frame shift mutation in
BY4741 resulting in an earlier stop codon and truncation of the protein,
which is assumed to be a loss-of-function gene product (Dimitrov et al.,
2009). PMS1 has an insertion of four amino acids at position 417 in
VR1-5B. The sequence of BY4741 in this interval is the same as that of
S288c 20, except for one nucleotide in SAL1 that causes an amino acid
change at position 131 (valine in BY4741 and methionine in S288c and
VR1-5B).

[0028] (C) Reciprocal hemizygosity analysis. For each gene in the
fine-mapped locus, two diploid strains were constructed in the
VR1-5B/BY4741 hybrid background that carried either the VR1-5B-derived
(left) or BY4741-derived (right) allele from the gene. The rest of the
genome was identical between the two hybrids. The reciprocal deletions
were engineered in the haploid strains, after which the proper haploids
were crossed to obtain the diploid hybrids. The ethanol tolerance of the
diploid hybrids was determined by scoring the growth of twofold dilutions
on 16% ethanol after 9 days. This revealed different contributions of the
parental alleles of MKT1, SWS2 and APJ1 to high ethanol tolerance.

[0029] FIG. 7: Effect of MKT1, SWS2 and APJ1 on ethanol tolerance.

[0030] (A) The ethanol tolerance of BY4741 (inferior wild type) and its
MKT1, SWS2 and APJ1 deletion strains was determined by scoring growth of
twofold dilutions on different ethanol concentrations.

[0031] (B) The ethanol tolerance of VR1-5B (superior wild type) and its
MKT1, SWS2 and APJ1 deletion strains was determined by scoring growth of
twofold dilutions on different ethanol concentrations.

[0032] (C) The MKT1-VR allele is beneficial for high ethanol tolerance.
MKT1-BY and MKT1-VR including 534 bp upstream and 344 bp downstream
regions of the ORF were cloned in the low-copy-number plasmid YCplac111
and expressed in BY4741 (BY1) and three segregants from VR1-5B/BY4741
that hold MKT1-BY (1D, 24A and 32B). The ethanol tolerance was determined
in two-fold dilutions on different concentrations of ethanol.

[0034] The ethanol tolerance of diploid single and double APJ1 deletion
strains was determined on YP medium with 10% ethanol (after 7 and 8 days)
and 18% ethanol (after 12 days) or with glucose (after 1 day) as control.

[0035] FIG. 9: Expression of APJ1 in BY4741 and VR1-5B strains during the
beginning of the fermentation.

[0036] Determination of APJ1 expression by Real-time PCR in BY4741 and
VR1-5B strains during the beginning of the fermentation showed a higher
expression level in the BY4741 strain (normalized to 1.0±0.19)
compared to the VR1-5B strain (0.43±0.12). This agrees with the
conclusion that Apj1p is negative for ethanol tolerance and that the APJ1
allele of VR 1-5B is superior because of its lower expression.

[0038] Ethanol tolerance of industrial yeast strains, Ethanol red (ER),
ES2 and PE2, and its single and double APJ1 deletion strains was
determined by scoring growth of twofold dilutions on nutrient plates with
different ethanol concentrations (12%, 14% and 16%). Industrial strains
with double APJ1 deletion showed a higher ethanol tolerance than the
control strains on YP plates with 16% ethanol.

[0039] FIG. 11: Comparison of fermentation performance in an industrial
xylose utilizing strain with wild type APJ1 and the same strain deleted
for APJ1 in both alleles.

[0040] Fermentation was performed in YP+35% (w/v) glucose, in continuous
stirring or static condition at 30° C. Values for stirring
fermentations are average of duplicate experiments. Fermentation tubes
were weighed every few hours and the amount of glucose leftover was
inferred from the weight loss due to CO2 release. (A) Fermentation
profile measured from weight loss due to CO2 release. (B) Final ethanol
level. (C) Final glucose leftover, and ethanol and glycerol produced. The
stirred fermentation has no indication, the static fermentation is
indicated as (static). GS1.11-26 is the parental strain, the double
mutant is indicated as GS1.11-26 APJ1ΔΔ.

[0047] Genomic DNA was extracted from yeast according to Hoffman and
Winston (1987). When required, additional purification was performed by
ethanol precipitation. Polymerase chain reaction (PCR) was performed with
Accuprime (Invitrogen) for cloning and sequencing purposes and with ExTaq
(TAKARA) for diagnostic purposes. Yeast was transformed with the LiAc/PEG
method (Gietz et al., 1995). Cloning was performed by standard
techniques. Dephosphorylation was performed with rAPid Alkaline
Phosphatase (Roche) and ligation with T4 DNA ligase (Roche). E. coli was
transformed with the CaCl2 method (Sambrooke et al., 1989) and
plasmid DNA isolated according to Del Sal et al., (1988).

[0048] Plasmid YCplac111 was described by Gietz and Sugino (1988).
YCplac111(MKT1-BY) and YCplac111(MKT1-VR) are the YCplac111 derivatives
carrying the MKT1 gene from BY4741 and VR-5B, respectively, pFA6-kanMX4
was described by Wach et al., (1994)

[0049] Mating, Sporulation and Tetrad Analysis

[0050] Mating, sporulation and tetrad analysis were performed by standard
procedures (Sherman and Hocks, 1991). The mating type of the segregants
was determined by diagnostic PCR for the MAT locus (Huxley et al., 1990).

[0051] Ethanol Tolerance Test

[0052] Strains were inoculated in YPD and grown at 30° C. for 3
days till stationary phase. Cultures were diluted to an OD600 of 0.5 and
5 μl of a twofold (100 till 8.10-3) or tenfold (100 till 10-3)
dilution range was spotted on YPD and YP with different concentrations of
ethanol. Growth was scored after one day for control YPD plates and 9 to
11 days for plates with ethanol. All spot tests were repeated at least
twice starting from independent cultures.

[0053] High Gravity Fermentations

[0054] Small scale fermentation was performed in 100 ml YP±35% (w/v)
glucose. Strains were first pre-grown in 5 ml YPD medium for about 24
hours at 30° C. The pre-culture was then transferred to 60 ml YPD
medium at an initial OD600 of 1. After the cells were allowed to grow to
stationary phase, they were harvested by centrifugation at 3000 rpm at
4° C. for 3 minutes, and the pellet was inoculated into 100 ml
fermentation medium. For stirring fermentation, continuous stirring was
applied at 120 rpm. For static fermentation, the cells were agitated only
for the first 4 hours. The glucose leftover was calculated from the
weight loss measurement resulted from CO2 evolution. Samples were taken
at the end of the fermentation and analyzed for the glucose leftover,
produced ethanol and glycerol by HPLC.

[0055] Genotyping of SNP Markers by PCR

[0056] For each SNP marker, two primers were constructed that differed
only at their 3' terminal end nucleotide. In particular, one primer
contained the VR1-5B nucleotide, while the other primer contained the
BY4741 nucleotide. Both primers were always applied in separate PCR
reactions with a common indirect primer. The two primer pairs were
investigated for their ability to specifically amplify the VR1-5B or
BY4741 sequence by performing four PCR reactions at different
hybridization temperatures that differed in the combination of DNA and
primer pairs. The combinations were: (1) DNA from BY4741 with primer pair
for BY4741, (2) DNA from BY4741 with primer pair for VR1-5B, (3) DNA from
VR1-5B with primer pair for BY4741 and (4) DNA from VR1-5B with primer
pair for VR1-5B. The PCR reactions were performed at hybridization
temperatures from 58° C. till 66° C. (2° C.
increments). The hybridization temperature at which the VR1-5B and BY4741
sequences were specifically amplified was subsequently applied to
genotype the SNP marker in individual highly ethanol tolerant segregants.
Each SNP marker check included VR1-5B and BY4741 as controls.

[0057] Real-Time PCR

[0058] For measurement of APJ1 expression, samples were taken from early
exponential-phase grown cells of BY4741 and VR1-5Ba. Pellets were frozen
in liquid nitrogen and stored at -80° C. RNA extraction was
performed using the phenol chloroform method. cDNA was prepared following
the instructions of the GOSCRIPT® Reverse Transcription System kit
(Promega). Relative quantification of APJ1 and 18S was performed using a
StepOnePlus Real-time PCR system (Applied Biosystems), primers: Fw APJ1
(TGATGGGCACGGTGGTCTA) (SEQ ID NO:5), Rv APJ1 (TTGAATACCTTGCCCTTT GCA)
(SEQ ID NO:6), Fw 18S (CACTTCTTAGAGGGACTATCGGTTTC) (SEQ ID NO:7) and Rv
18S (CAGAAC GTCTAAGGGCATCACA) (SEQ ID NO:8).

[0059] Preparation of DNA Samples for Whole-Genome Sequencing

[0060] The two parent strains VR1-5B and BY4741 and all segregants with
high ethanol tolerance were grown individually in 50 ml YPD at 30°
C. for 3 days. Exactly 10 ml of each culture was filtered, after which
the cells were dried in the microwave and weighed to establish the
relationship between optical density and dry weight. The remaining
culture volumes were stored at -80° C. The two pools of segregants
were constructed by combining equal amounts of cells from the stored
cultures based on dry weight. The genomic DNA from the parent strains and
the pools was extracted according to Johnston (1994). At least 3 μg of
each DNA sample was provided to GATC Biotech AG (Konstanz, Germany) for
sequencing.

[0061] Reciprocal Hemizygosity Analysis

[0062] All deletions for reciprocal hemizygosity analysis were made in the
haploid backgrounds. The BY4741 deletion strains were obtained from the
deletion strain collection (Giaever et al., 2002). The deletions in the
VR1-5B background were made using the same primers and strategy as the
International Deletion Consortium (Giaever et al., 2002; Winzeler et al.,
1999). The transformants were selected on geneticin plates and verified
by PCR with several combinations of internal and external primers. The
haploid strains were subsequently crossed to construct the diploid hybrid
strains. The presence of both the wild type and deletion allele of the
gene in the diploid hybrids was verified by PCR. The reciprocal
hemizygosity analysis was performed twice starting from independent PCR
amplifications and transformations.

[0063] Statistical Analysis

[0064] For every chromosome, the quantified frequencies of the detected
SNPs were considered to be binomially distributed. The underlying
structure in the SNP scatterplot of a given chromosome (FIG. 2) was
identified by fitting smoothing splines in the generalized linear mixed
model framework 48. The number of knots of the spline was chosen such
that they are spaced at approximately 40 kb intervals. Simultaneous
confidence bands 48 for the fitted smoother were constructed and allowed
identification of regions that are significantly different from a
baseline, i.e., a SNP frequency of 50%. For chromosome II and XV, the
data from both pools of segregants (16% and 17% ethanol) were
simultaneously modeled with generalized additive mixed models with a
smoother for the mean trend (FIG. 2B) and for the difference between both
pools. For graphical representation we have chosen to represent the
resulting fit for each pool and their simultaneous confidence bands. The
difference in SNP frequency between the two pools is certainly
significant when the simultaneous confidence bands do not overlap.

Example 1

Characterization of Parent Strains with High and Low Ethanol Tolerance

[0065] A segregant called VR1-5B was isolated from the Brazilian
bioethanol production strain VR1 that displayed similarly high ethanol
tolerance as the parent strain. Ethanol tolerance was thereby defined as
growth on solid YP plates with ethanol as the sole carbon source. Because
high ethanol tolerance is only relevant towards the end of yeast
fermentation when the sugar level has dropped to low values, ethanol
tolerance was determined in the absence of any other sugar or carbon
source. The VR1 parent strain could grow in medium containing up to 16%
ethanol, while the VR1-5B segregant showed growth in medium containing up
to 18% ethanol (FIG. 1). Both strains were clearly more ethanol tolerant
than the control haploid BY4741 and diploid BY laboratory strains, which
could grow only slightly in medium with 14% ethanol (FIG. 1). The diploid
VR1-5B/BY4741 strain displayed similarly high ethanol tolerance to the
VR1 parent strain, indicating that the high ethanol tolerance in this
strain is a dominant property (FIG. 1).

Example 2

Pooled-Segregant Whole Genome Sequence Analysis

[0066] From the cross between VR1-5B and BY4741, we obtained 5974
segregants that were phenotyped for ethanol tolerance by scoring growth
on YP with different concentrations of ethanol. The segregants with
extreme phenotypes were subsequently classified in two pools. The first
pool contained 136 segregants with a tolerance to at least 16% ethanol
(Pool 1) and the second pool contained 31 segregants from the first pool
with a tolerance to at least 17% ethanol (Pool 2). All segregants were
individually grown up till stationary phase, after which equal amounts of
cells based on dry weight were combined to obtain Pool 1 and Pool 2. The
genomic DNA from both pools and the parent strains was extracted and
submitted to custom sequence analysis using Illumina HiSeq 2000
technology (GATC Biotech AG, Konstanz, Germany). The sequencing was
performed at 40 times or greater coverage and generated paired-end short
reads of about 100 bp allowing a highly precise alignment of the reads.
The VR1-5B and BY4741 sequences were aligned to the reference S288c
genome sequence 20 and SNPs between VR1-5B and BY4741 with a coverage of
more than 20 times and a ratio of at least 80% were selected.
Subsequently, the sequence of the pool was aligned to the BY4741 sequence
and the nucleotide frequency of each SNP was plotted against its
chromosomal position. The SNP nucleotide frequency curve obtained by
whole-genome sequencing of DNA extracted from Pool 1 (16% ethanol)
fluctuated around 50% in most areas in the genome (FIG. 2A).

[0067] On the other hand, three loci showed a strong deviation from 50%
inheritance, containing SNPs with a frequency of less than 20% or higher
than 80% in the center of the locus. The loci were located on chromosomes
V, X and XIV. The significance of the deviation in SNP nucleotide
frequency could be confirmed by scoring a single SNP from the center of
each locus in at least 96 individual highly ethanol tolerant segregants
by PCR (Table 1). The QTLs on chromosome V (QTL1) and chromosome XIV
(QTL3) showed the strongest link, with respectively 92.8% and 94.1% of
the highly ethanol tolerant segregants harbouring the nucleotide from
VR1-5B. The locus on chromosome X (QTL2) showed a much weaker link, with
only 72.9% of the segregants showing VR1-5B inheritance. Scoring the same
SNPs in an unselected pool of at least 80 segregants resulted in an
association percentage of 50.0%, which is consistent with random
segregation of the QTLs in an unselected pool of segregants. The joint
effect of the three QTLs on high ethanol tolerance was examined by
determining the appearance of each of the eight combinations in 85 highly
ethanol tolerant segregants (Table 2). The combination between the
VR1-5B-derived alleles from QTL1 and QTL3 was most prevalent in the
segregants. Taken together, 88.2% of the highly ethanol tolerant
segregants carried the VR1-5B-derived alleles from QTL1 and QTL3,
indicating that inheriting both alleles is strongly advantageous for high
ethanol tolerance. These results revealed that the VR1-5B-derived alleles
from QTL1 and QTL3 are the major contributors to the high ethanol
tolerance phenotype and that QTL2 is less important. The three identified
QTLs were confirmed by whole-genome sequence analysis of DNA extracted
from Pool 2 (17% ethanol) (FIG. 3). These data also revealed significant
deviations from 50% inheritance at several other loci, which appear to
represent minor loci determining high ethanol tolerance (FIG. 4). For
example, a locus on chromosome II and on chromosome XV did not show a
clear deviation from random segregation in the pool of segregants
tolerant to 16% ethanol, whereas a clear deviation was observed in the
pool of segregants tolerant to 17% ethanol (FIG. 2B). The boundaries of
the two major loci (QTL1 and QTL3) identified in both pools by
pooled-segregant whole genome sequence analysis were determined by
scoring selected SNP markers in the region of the locus for at least 68
individual segregants that composed Pool 1 (16% ethanol) by PCR. We
calculated the P value for each SNP using an exact binomial test with a
confidence level of 95% and correction for multiple testing by a false
discovery rate (FDR) control according to Benjamini-Yekutieli (2005). The
P values were plotted over the length of the chromosome for each
identified locus (FIG. 5).

Example 3

Genetic Dissection of QTL3 Reveals Two Positive and One Negative Genetic
Element

[0068] The 370 kb QTL3 was fine-mapped using selected SNPs to reduce the
size of the interval to a practical number of candidate genes for further
functional analysis. The P values for eight SNP markers (S67, S67-1,
S67-2, S68, S68-1, S68-2, S68-3, S69) defined a smaller locus of 16 kb
between markers S68 and S68-2, which had the strongest link (FIG. 6A).
The locus contained ten annotated genes (FIG. 6B). Sanger sequence
analysis of this region was performed to detect all nucleotide
polymorphisms between VR1-5B and BY4741 (FIG. 6B). We observed that
VR1-5B and BY4741 were highly divergent with a polymorphism on average
every 167 bp. All genes except TPM1 had at least one polymorphism in
their ORF, being silent mutations for the genes APJ1 and SWS2 and
missense mutations in the other seven genes. In addition, all genes had
at least one polymorphism in their putative promotor and/or terminator.
Given the difficulty to predict the effect of both coding and non-coding
polymorphisms on phenotypes (Tabor et al., 2002), the sequence data could
not be used to exclude genes from further functional analysis. Reciprocal
hemizygosity analysis (RHA) was applied to identify the causative genes
in the locus. RHA allows analyzing whether the two parental alleles have
a different contribution to the phenotype in an otherwise uniform genetic
background (Steinmetz et al., 2002). For nine genes, two heterozygous
strains were constructed in the VR1-5B/BY4741 hybrid background that only
differed genetically in the candidate gene, i.e., they carried either one
copy of the VR1-5B or the BY4741 allele while the other copy of the gene
was deleted (FIG. 6C). Comparing the ethanol tolerance of each pair of
heterozygous strains revealed a difference in the phenotypic contribution
between the parental alleles of MKT1, SWS2 and APJ1 (FIG. 6D). The
presence of the VR1-5B allele of the MKT1 and APJ1 gene resulted in
higher ethanol tolerance compared to the BY4741 allele. For SWS2 the
opposite was true, as the BY4741 allele was advantageous over the VR1-5B
allele. One potential complication with RHA is that the hybrid diploid
background used in the assay is different from the haploid segregants
background used in the QTL mapping experiment. For this reason, we
determined the deletion phenotypes of MKT1, SWS2 and APJ1 in the VR1-5B
and BY4741 haploid strains. In the BY4741 background (which has a much
lower ethanol tolerance), the MKT1Δ strain showed only a minor
growth reduction while the APJ1Δ strain grew equally well as the
wild type strain on 10% ethanol (FIG. 7A). Similar results were obtained
on 12%, 14%, 15% and 16% ethanol (FIG. 7A). In contrast, deletion of SWS2
resulted in complete loss of growth on all ethanol levels (FIG. 7A).
These results are in agreement with those of the screening of the BY
deletion strain collection that only observed an ethanol sensitive growth
phenotype for the sws2Δ strain. In the VR1-5B background (which has
a much higher ethanol tolerance), deletion of SWS2 but also of MKT1,
caused a severe growth defect on 10%, 12%, 14%, 15%, 16%, 17%, 18% and
19% ethanol (FIG. 7B). Interestingly, deletion of APJ1 had no effect for
growth on 10% ethanol, but caused a clear growth improvement on 12%, 14%,
15% and 16% ethanol (FIG. 7A). Similar results were obtained for deletion
of APJ1 in the VR1-5B background, but the positive effect on the higher
ethanol levels was smaller than in the BY4741 background (FIG. 7B). The
improvement of ethanol tolerance by deletion of APJ1 indicates that the
APJ1 gene product negatively affects ethanol tolerance. When this is
combined with the result of the RHA analysis, it suggests that the
beneficial effect on ethanol tolerance of the APJ1-VR allele is due to
lower expression compared to that of the APJ1-BY allele. The relevance of
MKT1 for high ethanol tolerance was confirmed by expressing both parental
alleles in BY4741 and in segregants from VR1-5B/BY4741 that hold the
BY4741-derived allele of MKT1. Expression of MKT1-VR in contrast to
MKT1-BY resulted in higher ethanol tolerance in BY4741 and two out of the
three segregants (FIG. 7C). This confirmed the result from RHA suggesting
that MKT1-VR is advantageous for high ethanol tolerance. On the other
hand, as we did not observe an effect in all segregants, it seems that
MKT1 alone is not sufficient to enhance ethanol tolerance. Comparing
ethanol tolerance in the strains BY4741 and BY4741mkt1Δ confirmed
that MKT1-BY is a loss-of-function allele, since no difference in ethanol
tolerance was observed (FIG. 7C). In contrast, deletion of MKT1 in VR1-5B
lowered ethanol tolerance (FIG. 7C), which confirms that a loss-of
function mutation in MKT1 decreases ethanol tolerance.

[0069] Also the single and double deletion of ARJ1 in diploid strains
improved ethanol tolerance. This was observed with the diploid
VR1-B/VR1-B single and double APJ1 deletion strains and with the diploid
BY4741/BY4741 double APJ1 deletion strain (FIG. 8).

[0070] The role of APJ1 is further confirmed by analysis of APJ1
expression in BY4741 and VR1-5B, using real time PCR (FIG. 9). VR1-5B
shows a lower APJ1 expression and a higher ethanol resistance, supporting
the idea that a high APJ1 expression is negative for ethanol tolerance.

[0071] Single and double APJ1 deletion mutants were made from the diploid
industrial yeast strains Ethanol red (ER), ES2 and PE2. The ethanol
tolerance of the parental strains and of the single and double deletion
mutants was scored by growth on YP plates with increasing ethanol
concentration (10 days incubation, 12%, 14%, and 16% ethanol). Growth on
YPD after 1 day incubation was used as control. The results are shown in
FIG. 10. No difference in growth could be noticed for the control; all
double mutants scored clearly better in ethanol tolerance.

[0072] The ethanol tolerance inducing effect of the APJ1 deletion was
further confirmed in high gravity fermentation. Both APJ1 alleles were
deleted in the xylose utilizing ER derivative GS1.11-26 strain. The
resulting double deletion strain was used in static and stirred high
gravity fermentation in a YP medium comprising 35% glucose. The results
are summarized in FIG. 11. The double mutants were faster fermenting both
in static and in stirred conditions. No clear difference in final ethanol
production and glucose consumption could be seen in stirred conditions,
but the double deletion mutant performed clearly better (higher ethanol
production and glucose consumption at the end of the fermentation).

TABLE-US-00001
TABLE 1
Statistical confirmation of the significance
of the three identified QTLs.
Name Position of SNP Number SNP frequency P value
QTL1 chr V; 122,599 125 92.8% <<1.0E-09
QTL2 chr X; 659,775 96 72.9% .sup. 8.1E-06
QTL3 chr XIV; 468,914 101 94.1% <<1.0E-09
An SNP in the middle of each QTL was scored in at least 96 individual
highly ethanol tolerant segregants by PCR using specific primers for the
two alleles. The P values were calculated with a confidence level of 95%.